Metabolic characterisation of the nutritional versatile marine bacterium Phaeobacter inhibens DSM 17395 via gas chromatography – mass spectrometry Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo Wilhelmina zu Braunschweig zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Michael Hensler aus Lüdenscheid 1. Referent: Professor Dr. Dietmar Schomburg 2. Referent: Professor Dr. Ralf Rabus 3. Referent: Professor Dr. Dieter Jahn eingereicht am: 24.11.2014 mündliche Prüfung (Disputation) am: 05.03.2015 Druckjahr 2015 Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Publikationen Zech, H., Hensler, M., Koßmehl, S., Drüppel, K., Wöhlbrand, L., Trautwein, K. Hulsch, R., Maschmann, U., Colby, T., Schmidt, J., Reinhardt, R., Schmidt-Hohagen, K., Schomburg, D., Rabus, R. (2013). Adaptation of Phaeobacter inhibens DSM 17395 to growth with complex nutrients. PROTEOMICS 13, 2851-2868. Zech, H., Hensler, M., Koßmehl, S., Drüppel, K., Wöhlbrand, L., Trautwein, K., Colby, T., Schmidt, J., Reinhardt, R., Schmidt-Hohagen, K., Schomburg, D., Rabus, R. (2013). Dynamics of amino acid utilization in Phaeobacter inhibens DSM 17395. PROTEOMICS 13, 2869-2885. Drüppel, K., Hensler, M., Trautwein, K., Koßmehl, S., Wöhlbrand, L. Schmidt-Hohagen, K., Ulbrich, M., Bergen, N., Meier-Kolthoff, J.P., Göker, M., Klenk, H.P., Schomburg, D., Rabus, R. (2014). Pathways and substrate-specific regulation of amino acid degradation in Phaeobacter inhibens DSM 17395 (archetype of the marine Roseobacter clade). Environ. Microbiol. 16, 218-238. Wiegmann, K., Hensler M., Wöhlbrand, L., Ulbrich, M., Schomburg, D., Rabus, R. (2014). Carbohydrate Catabolism in Phaeobacter inhibens DSM 17395, Member of the Marine Roseobacter Clade. Appl. Environ. Microbiol. 80, 4725-4737. Tagungsbeiträge (Vorträge) Hensler, M., Bill, N., Rex, R. (2011). Metabolome analysis and modelling of the metabolisms of Dinoroseobacter shibae and Phaeobacter gallaeciensis. 3. Statusseminar des SFB TRR 51, 12. – 13.05.2011, Delmenhorst. Hensler, M. (2013). Adaptation of Phaeobacter inhibens DSM 17395 to nutrient conditions in marine environments. International Symposium of SFB TRR 51, 24. – 26.06.2013, Delmenhorst. Wiegmann, K., Hensler, M. (2014). Carbohydrate catabolism in Phaeobacter inhibens DSM 17395 – Proteomic and Metabolomic analyses. 8. Statusseminar des SFB TRR 51, 08. – 09.05.2014, Oldenburg. Posterbeiträge Hensler, M., Zech, H., Koßmehl, S., Drüppel, K., Wöhlbrand, L., Trautwein, K., Schmidt- Hohagen, K., Rabus, R., Schomburg, D. (2013). Dynamics of amino acid utilization by Phaeobacter gallaeciensis DSM 17395. Internationale Konferenz der VAAM, 10. – 13.03.2013, Bremen. Hensler, M., Zech, H., Drüppel, K., Koßmehl, S., Wöhlbrand, L., Trautwein, K., Schmidt- Hohagen, K., Rabus, R., Schomburg, D. (2013). Metabolic and proteomic adaptations of Phaeobacter inhibens DSM 17395 to nutrient conditions in marine environment. Metabolomics 2013 – 9th Annual Conference of the Metabolomics Society, 01. – 04.07.2013, Glasgow, United Kingdom. Hensler, M., Wiegmann, K., Zech, H., Koßmehl, S., Rabus, R., Schomburg, D. (2014). The metabolism of the marine all-rounder Phaeobacter inhibens DSM 17395. Trends in Metabolomics – Analytics and Applications. 03. – 05.06.2014, Frankfurt am Main. TABLE OF CONTENTS I Table of Contents List of figures.....................................................................................................................VI List of tables...................................................................................................................VIII Abbreviations.....................................................................................................................IX Summary............................................................................................................................XI Zusammenfassung...........................................................................................................XII 1 Introduction.......................................................................................................................1 1.1 Marine environment as habitat (for bacteria)..............................................................1 1.1.1 Characteristics of seawater..................................................................................1 1.1.2 Dissolved organic matter.....................................................................................3 1.1.3 Seasonal and regional variations of dissolved organic matter.............................5 1.2 The Roseobacter clade and Phaeobacter inhibens......................................................6 1.2.1 The Roseobacter clade.........................................................................................6 1.2.2 The genus Phaeobacter and the strain P. inhibens DSM 17395..........................8 1.3 Systems biology.........................................................................................................10 1.4 Metabolomic analyses................................................................................................11 1.4.1 Introduction to Metabolomics............................................................................11 1.4.2 Sample preparation techniques for bacterial metabolomics..............................12 1.4.3 Gas chromatography – mass spectrometry........................................................13 1.4.4 Liquid chromatography – mass spectrometry....................................................14 1.5 Objectives..................................................................................................................15 2 Material and methods.....................................................................................................16 2.1 Equipment and instruments.......................................................................................16 2.2 Consumables..............................................................................................................17 2.3 Chemicals..................................................................................................................18 II TABLE OF CONTENTS 2.4 Organism....................................................................................................................18 2.5 Media and supplements.............................................................................................19 2.5.1 Salt water medium (minimal medium)..............................................................19 2.5.2 Bacto Marine Broth (complex medium)............................................................21 2.5.3 Biolog-inoculum solutions.................................................................................21 2.5.4 Solutions for GC-MS analysis...........................................................................21 2.6 Software.....................................................................................................................22 2.7 Databases...................................................................................................................23 2.8 Microbial techniques.................................................................................................24 2.8.1 Biolog phenotypic microarray analysis.............................................................24 2.8.2 Isotope labelling.................................................................................................24 2.8.3 Study of coenzyme A derivatives linked with tryptophan degradation.............25 2.8.4 Cultivation conditions for main experiments.....................................................25 2.9 Proteomic analyses....................................................................................................26 2.10 Analysis of coenzyme A derivatives........................................................................26 2.10.1 Extraction of coenzyme A derivatives.............................................................26 2.10.2 Liquid chromatography – mass spectrometry analysis....................................27 2.10.3 Data processing and peak identification..........................................................28 2.11 Sample preparation for GC-MS analysis.................................................................29 2.11.1 Extraction of intracellular metabolites.............................................................29 2.11.2 Preparation of culture supernatants..................................................................30 2.11.3 Derivatisation reaction.....................................................................................30 2.12 Gas chromatography – mass spectrometry analysis................................................30 2.12.1 Leco Pegasus 4 d GC × GC TOF MS..............................................................31 2.12.2 Jeol JMS-T100GC Accu TOF MS...................................................................31 2.12.3 DSQ II GC-MS................................................................................................32 2.13 Setup of main experiments......................................................................................32 2.13.1 Adaptation to growth with complex medium...................................................33 2.13.2 Dynamics of